The invention relates to compositions and the methods for use in improving the sensitivity and specificity of polynucleotide synthesis. Specifically, the [inventive] method comprises protecting the 3xe2x80x2-end of an oligonucleotide used as a primer in the synthesis of a polynucleotide to prevent non-specific chain elongation [reaction], and removing the protection at an elevated temperature, using a thermostable enzyme, to allow template-specific polynucleotide synthesis. The present invention is also directed to a thermostable 3xe2x80x2 polynucleotide phosphatase and its use as a marker protein. The present invention further relates to oligonucleotides with a 3xe2x80x2 end protected by a blocking group. The instant invention also relates to methods for improving the sensitivity and specificity of amplification and analysis of DNA/RNA from a variety of samples. The compositions and methods of the invention are very useful in a variety of techniques for DNA/RNA amplification and analysis, including medical genetics research and diagnosis, pathogen detection, forensic, and animal and plant genetics applications, among others.
Polynucleotide synthesis is a process of information transfer and usually requires another polynucleotide molecule as a template. In general, template-dependent polynucleotide synthesis involves the denaturing of the template polynucleotide molecule, the annealing of a primer molecule, and a step of chain extension whereby the 3xe2x80x2 terminus of the primer is extended by a polynucleotide polymerase, using nucleoside-5xe2x80x2-triphosphate. This process is often repeated many times in vitro, such as in the case of a polymerase chain reaction (PCR).
Even though this information transfer process is remarkably accurate, the biomolecular machinery involved in the information transfer is not error-proof. These errors are a source of mutation in nature, and pose significant problems of infidelity for in vitro reactions such as PCR, random primer labeling, DNA sequencing, and reverse transcription. Both binding of primers to nonhomologous sites (xe2x80x9cmisprimingxe2x80x9d) and incorporation by the polynucleotide polymerase of incorrectly paired bases in the chain extension step are causes of the errors.
The availability of thermostable DNA polymerases which are stable at a temperature of up to 95xc2x0 C., such as the Taq DNA polymerase isolated from the thermophilic bacterium Thermus aquaticus and Pfu DNA polymerase from the thermophilic archaebacterium Pyrococcus furiosus has improved the specificity and sensitivity of PCR by significantly reducing mispriming.
The isolation of Tth polymerase, a thermostable polymerase from Thermus thermophilus (Tth polymerase), that can function as both reverse transcriptase and DNA polymerase (Myers and Gelfand, Biochemistry 30:7662-7666 (1991)), has overcome the limitation of mesophilic viral reverse transcriptases which can only function at lower temperatures and which cannot xe2x80x9cread throughxe2x80x9d the secondary structures of the RNA template at the low temperature. The reverse transcription performed at an elevated temperature using Tth polymerase eliminates secondary structures of template RNA, making the synthesis of full-length cDNA possible.
In most uses of PCR, the primer should bind very specifically to the target sites. While primer binding usually is very specific at elevated temperatures, the reaction mixture[,] must be held at lower temperatures (such as the ambient temperature) at certain stages of the PCR process, especially during the assembly of the reaction cocktail prior to the PCR temperature cycle. At lower temperatures, the primers may undesirably bind to the non-targeted nucleic acids, or to other primer molecules in the reaction mixture, resulting in nonspecific primer extension products and primer dimers, in addition to the specific product produced from the target nucleic acid. These undesired products cause high background, decrease amplification efficiency, and lower reaction specificity.
Despite recent progress in PCR technology, mispriming of background DNA and primer oligomerization still present a significant problem. This is especially true in diagnostic applications in which PCR is carried out in a milieu in which there are only a few copies of the target DNA (Chou et al. Nucleic Acid Res. 20:1717-1723 (1992)). It has been determined that non-specific chain extension by the DNA polymerase often occurs when all reactants have been mixed at ambient temperature, before thermal cycling is initiated, resulting in undesirable spurious amplification products.
Three methods have been reported which minimize these side reactions. The first method, termed xe2x80x9chot startxe2x80x9d PCR, has various permutations, (Chou et al. Nucleic Acid Res. 20:1717-1723. (1992); D""Aquila et al. Nucleic Acid Res. 19:3749(1991)), with the common feature that all of the reagents are heated to 72xc2x0 C. before a final reagent, usually the polymerase, is added to the reaction cocktail, preventing mispriming and primer oligomerization. Although this method does increase specificity, thereby reducing side products, the method is error-prone and tedious for dealing with a large number of samples, and the reaction mixture can become more easily contaminated.
In the second method, a polymerase-neutralizing antibody, for example, the Taq polymerase antibody sold under the tradename TaqStart Antibody, is added to the complete reaction mixture. This antibody inhibits the polymerase activity at ambient temperature (Kellogg et al. Biotechniques 16:1134-1137 (1994)), but is inactivated by heat denaturation once the reaction is thermocycled, releasing the active polymerase. The drawback of this approach is that the antibody needs to be stored at xe2x88x9220xc2x0 C. until use, which means that detection kits need to be packaged and shipped in a controlled environment, adding to their cost. In addition, a significant amount of antibody (about 1 xcexcg of antibody/5 U of Taq) is needed for a single PCR. The added antibody represents a significant amount of protein in the reaction mixture and interferes with further analysis of the PCR products by immunochemical assays such as ELISA.
In the third method, a modified form of Taq DNA polymerse, named AmpliTaq Gold, is employed in the PCR (Birch, D. E. et al. Nature 381:445-6 (1996)). The AmpliTaq Gold is inactive at room temperate and has to be heated to a temperature above 90xc2x0 C. for at least 5 minutes in order to restore its activity. Therefore, a pre-PCR heating step at 95xc2x0 C. is required. Although the AmpliTaq Gold can be activated during cycling and pre-PCR heat step can be eliminated, ten or more extra cycles are necessary to give equivalent product yield. Furthermore, many researchers find that it is difficult to amplify certain length of target sequence, for example 4 kb, from human genomic DNA by using AmpliTaq Gold.
There is therefore a need in biotechnology and molecular biology for an improved method for improved specificity in polynucleotide polymerization with reduced mispriming and primer oligomerization.
Moreover, there is also a great need in biotechnology and molecular biology for markers that can be used to study gene expression, regulation and function. For example, the functional genomics project requires that the expression pattern of newly discovered genes be determined. Currently, several methods are available to monitor gene regulation and expression. These include the formation of fusion proteins with coding sequences for xcex2-galactosidase and luciferases (Reviewed in T. J. Silhavy and J. R. Beckwith, Microbiol. Rev. 49:398 (1985); S. J. Gould and S. Subramani, Anal. Biochem. 175:5 (1988); and G. S. A. B. Stewart and P. Williams, J. Gen. Microbiol., 138:1289 (1992)). Another protein that has been extensively used as a reporting marker in this field is the green fluorescent protein (reviewed by Misteli and Spector, Nat Biotechnol. 15(10):961-4 (1997); Cormack, Curr Opin Microbiol 1(4):406-10 (1998)). However, these enzymes are temperature sensitive and their assay have to be carried out in a short time period after sample preparation.
Similarly, current method utilizing these or similar enzymes for the determination of the presence and concentration of certain ligands also suffer the drawbacks discussed above, and there is a need for new methods utilizing enzymes whose activities are easily to assay and do not deteriorate rapidly at elevated temperatures.
It is an object of the instant invention to provide a composition comprising an oligonucleotide primer which has a 3xe2x80x2 terminal hydroxy that is protected by a blocking-group. The 3xe2x80x2-end modified primer, according to the instant invention, prevents the chain-extension reaction by a DNA polymerase or a reverse transcriptase. Chain extension is even inhibited in the presence of all the necessary reaction components including dNTPs and metal cofactors.
The instant invention further provides a method for the addition of a nucleotide to a 3xe2x80x2-hydroxyl terminus of a preexisting polynucleotide molecule, comprising: (A). contacting a specimen suspected of containing a target polynucleotide molecule with a reaction mixture comprising (1) a primer complementary to said target polynucleotide molecule, wherein the 3xe2x80x2-hydroxyl terminus of the primer is protected with a blocking group that prevents chain-elongation; (2) a thermostable blocking group removing enzyme (TBGRE); (3) a thermostable polynucleotide polymerase, (3) at least one nucleoside-5xe2x80x2-triphosphate; and (B). elevating the temperature of the resulting mixture to higher than about 38xc2x0 C., thereby generating a primer extension product. In a preferred embodiment, the instant method further comprises a step of (C) denaturing the primer extension product and conducting at least one additional primer extension reaction.
It is another object of the invention to provide a purified or recombinant thermostable 3xe2x80x2-polynucleotide phosphatase. The thermostable 3xe2x80x2-polynucleotide phosphatase according to the instant invention is capable of removing a 3xe2x80x2 phosphate moiety of a 3xe2x80x2 end modified primer only at a sufficiently high temperature. The removal of the 3xe2x80x2 phosphate moiety allows the chain extension reaction to commence and continue only when the temperature of the reaction cocktail is sufficiently high, thus preventing non-specific amplification. Still further provided by the instant invention is a recombinant thermostable 3xe2x80x2 polynucleotide phosphatase from Pfu.
This invention also relates to a method for producing a recombinant thermostable 3xe2x80x2 polynucleotide phosphatase that is derived from a hyperthermophilic archaebacteriumm P. furiosus 
The present invention also relates to using the thermostable 3xe2x80x2-phosphatase as a marker protein. For example, this invention relates to a method for quickly determining whether an in vitro expression system successfully expresses the target protein, the method comprising (1) fusing a DNA molecule having DNA sequence encoding the protein of interest to the DNA sequence encoding a thermostable 3xe2x80x2 phosphatase activity such that the protein produced by the DNA molecule will have the protein of interest fused to the 3xe2x80x2 phosphatase of the invention; (2) placing the fusion gene in a suitable expression vector; (3) expressing the fused-DNA molecule; (4) heating the expressing cells to a temperature of 70xc2x0 C.; and (5) detecting the thermostable 3xe2x80x2-phosphatase activity.